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OPEN Skin gland concentrations adapted to diferent evolutionary pressures in the head and posterior regions of Received: 28 November 2017 Accepted: 14 February 2018 the caecilian Siphonops annulatus Published: xx xx xxxx Carlos Jared1, Pedro Luiz Mailho-Fontana1, Rafael Marques-Porto1, Juliana Mozer Sciani1, Daniel Carvalho Pimenta 1, Edmund D. Brodie Jr.2 & Marta Maria Antoniazzi1

Amphibian skin is rich in mucous glands and poison glands, secreting substances important for gas exchange and playing a fundamental role in chemical defense against predators and microorganisms. In the caecilian Siphonops annulatus (Mikan, 1920) we observed a concentration of enlarged mucous glands in the head region. In the posterior region of the body a similar concentration is made up of enlarged poison glands. These accumulations of glands structurally resemble the macroglands previously reported in anurans and salamanders. The skin glands in these regions are each surrounded by collagen walls forming a honeycomb-like structure. The collagen network in the head region frmly attaches to tiny pits in the bones of the skull. The two extremities of the body produce diferent secretions, containing exclusive molecules. Considering the fossorial lifestyle of caecilians, it seems evident that the secretions of the head and caudal region serve diferent functions. The anterior macrogland of mucous glands, rich in mucous/lipid secretion, in conjunction with the funnel-shaped head, may act to lubricate the body and penetrate the soil, thus facilitating locomotion underground. The blunt posterior end bearing an internalized macrogland of poison glands in the dermis may act in chemical defense and/or by blocking invasion of tunnels.

Caecilians are limbless amphibians comprising the Order Gymnophiona that are distributed in Southeast Asia, Central and South America and Africa1–3. Tey are fossorial animals, with compact skull, reduced visual system and a pair of sensory tentacles2–5. Possibly because of their fossorial habits and tropical distribution, caecilians constitute one of the least studied groups of vertebrates6. Tere are only 206 species (less than 3% of total extant amphibians) distributed in 10 families7. As in other amphibians, caecilian skin is rich in glands which secrete substances that are fundamental to sev- eral vital functions, including chemical defense against predators and microorganisms1,5,8,9. Among amphibians, two basic types of cutaneous glands are present: mucous glands and poison glands. Te mucous glands, in the form of typical acini, contain a characteristic lumen and secrete hydrophilic mucus that keeps the skin moist, facilitating gas exchanges1,5,9,10. Te poison glands have no lumen and store their toxins in the form of granules (hence their traditional designation as granular glands)9,11,12. Unlike the orders Anura and Urodela, Gymnophiona skin does not show any apparent glandular accumu- lations (macroglands), such as the parotoids, typically found in toads and salamanders and usually related to defense against predators5,13–16. It has long been known that granular glands are enlarged and most numerous in the posterior region of caecilians17,18. In this study we analyze the skin morphology and the biochemical compo- sition of the cutaneous secretion of diferent regions of the body of the caecilian Siphonops annulatus (Fig. S1), a species widely distributed in South America19. Although this caecilian has a homogeneous body surface without protuberances, its head and posterior regions exhibit glandular accumulations in the dermis. In the head region the accumulation is comprised of mucous glands, whereas in the posterior region, it is comprised of poison glands. Biochemical diferences also refect specializations between secretions extracted from the two regions. Taking into account the biology and natural history of Siphonops annulatus, we speculate about the role of glandular accumulations in this species and suggest that the composition of the cutaneous secretion in the

1Instituto Butantan, São Paulo, Brazil. 2Utah State University, Logan, UT, USA. Correspondence and requests for materials should be addressed to E.D.B. (email: [email protected])

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Figure 1. Internal morphology of the head skin and anatomy of the skull of Siphonops annulatus. (a) Internal aspect of the dermis of the snout tip revealed in transversal section, as exemplifed in the insert. Note the large number of skin glands arranged side by side. (b) Higher magnifcation of (A) revealing the predominance of mucous glands (mu), which are identifed by the presence of lumens. Note the collagen walls among the glands (*), conferring to the dermis a honeycomb appearance. (c) Image of a corresponding region of (b) afer removal of the mucous glands, leaving only the collagen. (d–e) Te skull of S. annulatus show many tiny orifcies (arrows) mainly in the nasopremaxilla (np), maxillopalatine (m), frontal (f), squamosal (sq), parietal (p) and pseudodentary bones (pd). Eye (e), tentacle (t). Scanning electron microscopy (a and b), stereomicroscopy (a insert, c–e).

head and posterior regions is related, respectively, to locomotion and defense against predators in the fossorial environment. Results Morphology. Te bluish-gray skin of Siphonops annulatus is smooth and shiny with well-developed annuli. In spite of the annuli, the surface of the body is homogeneous and, apart from the pair of tentacles, there are no visible protrusions. However, when the skin of the head and the posterior region is tangentially sectioned, a large number of densely packed glands are revealed within the dermis, forming honeycomb-like structures with dis- tinctly diferent morphological characteristics at each end of the body (Figs 1 and 2). Each unit of the honeycomb is formed by collagen walls, surrounding each gland, either mucous or poison gland. At the head, the honeycomb structure exclusively contains large mucous glands identifed by the presence of a characteristic central lumen, usually containing secretion (Fig. 1a,b). In contrast, at the posterior region, the honeycomb structure contains poison glands characterized by secretory cells completely flled with spherical granules and the absence of a lumen (Fig. 2a,b). Both in the head and in posterior region, afer gland removal, the dermal structure reveals the collagen walls composing the honeycomb/glands arrangement (Figs 1c and 2c). Te collagen of the walls in the posterior region is much thicker (Fig. 2d) but more pliable than that of the head. Afer total removal of the skin from the head, the skull of S. annulatus reveals that the bones form a single compact and robust structure (Fig. 1d) with a large number of tiny orifces, mainly distributed in the bones of the frontal, superior and lateral portions of the skull (Fig. 1d,e). Te connective tissue matrix forming the honeycomb structure surrounding the glands extend into the pits of the skull anchoring the skin to the skull (Fig. 3a). Microscopic evaluation of the skin reveals two types of mucous glands, Type I and Type II (Fig. S2), in addi- tion to poison glands. Sections of the head reveal an accumulation of Type I mucous glands of much larger dimensions than those found in the rest of the body (Fig. 3a−f). Histological analysis of skin sections show that, in progression from anterior to posterior, Type I mucous glands progressively decrease in number and size (Fig. 3a–f), whereas poison glands become more numerous and larger (Fig. 3a–f). Specifcally, toward the cloacal region, the poison glands are much enlarged (about 2.2 mm in height), occupying practically the entire volume of the dermis (Fig. 3f). Type II mucous glands are homogeneously distributed throughout the body, with the clear exception of the head (Fig. 3a–f).

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Figure 2. Anatomy of the skin of the posterior region of Siphonops annulatus. (a) Tangential section through the skin of the posterior tip of the body, just afer the cloaca (cl), exposing a great number of large glands in the dermis. (b) Higher magnifcation of (a) revealing the poison glands (g), predominant in this region. Note the absence of lumens in the glands and the thick collagen walls (*) between glands. Te insert shows a higher magnifcation of the poison stored in the glands in the form of granules. (c) Te honeycomb architecture of the skin becomes more evident afer removal of the poison from the glands. Note the presence of many pores in the skin surface (arrows), each one corresponding to a gland. (d) Higher magnifcation of the delimited region in (c) showing the honeycomb arrangement and the collagen walls (*), which remain unchanged afer poison removal. Scanning electron microscopy (a−b), stereomicroscopy (c−d).

Te secretory epithelia of Type I and Type II mucous glands are composed of two cell types, which stain at diferent levels of afnity to PAS (Fig. S3A–B) and alcian blue (Fig. S3C). Te two types of mucous glands stain poorly with bromophenol blue (Fig. S3D). In Type I glands, all cells of the secretory epithelia entirely stain with Sudan black, while in Type II glands only one kind of cell is strongly stained (Fig. S3E–B). Te morphological characteristics of poison glands, despite their variation in size, remain constant through- out the skin. Tey are always larger than the mucous glands and are also composed of two cell types (Figs S2A and S3D). One of these cell types is always located in an upper position in the gland, just below the duct (Fig. S3A), and has granules exclusively positive to PAS (Fig. S3B). Te remainder of the glandular body is composed of cells full of granules that strongly react to bromophenol blue (Fig. S3D).

Biochemistry. Te cutaneous secretion extracted from the head is viscous, colorless, and transparent, while that extracted from the posterior region is more fuid, milky and opaque. Te abundance of secretory proteins extracted from the posterior portion (11 mg/ml) is far greater than that from the head (0.2 mg/ml). Electrophoretic profles of secretions extracted from the head and from the posterior region show many difer- ences in all regions of the gel, with several exclusive bands (Fig. 4a, Fig. 4S). Chromatographic profles reveal sig- nifcant diferences between the head and posterior secretions, reinforcing their disparity. In the posterior region the secretion shows a majority peak in the polar region of the chromatogram (Fig. 4b). In addition, quantitative diferences are found between the two secretions, which are most obvious at peaks eluted between 0 to 1 min and 8 to 22 min (Fig. 4b insert). Discussion Among the amphibian orders, Gymnophiona is undoubtedly the least studied in all biological aspects. Skin mor- phology of caecilians remains practically unexplored despite marked and curious peculiarities compared to that of anurans and urodeles. Several of these peculiarities have been described in isolation but have never been ana- lyzed in an integrative context. Some interesting caecilian skin features have been observed since the nineteenth century20. Following Sarasin and Sarasin20 and Ochoterena21, Sawaya22,23 observed heterogeneous distribution of glandular secretions and lethality in Siphonops annulatus. Sawaya23 experimentally demonstrated the poison of this species, both the mucous (extracted from the head) and the granular (extracted from the posterior region), is toxic to the caecilian itself, the toad (Rhinella icterica), the frog (Leptodactylus ocellatus) and the rat (Rattus

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Figure 3. Characteristics and distribution of the cutaneous glands along the body of Siphonops annulatus. Te letters (a−f) in the diagram, refer to the diferent regions of the skin analysed in the histological fgures and correlate with the panels presented. (a) Sagittal section of the head. Note the predominance of Type I (I) mucous glands and the anchor spots (*) of the dermis (d) to the nasopremaxilla (np) bone. (b) Section of the skin, just posterior the head, showing abundance of Type I mucous glands (I). (c) Skin of the central-proximal region of the body, where the poison glands (g) and Type II mucous glands (II) are most frequent. (d−f) Moving posteriorly toward the body terminus, the poison glands (g) become progressively larger and more abundant. Epidermis (e). Stain: haematoxylin-eosin (a−f).

norvegicus). Characteristics such as the heterogeneous distribution of poison and mucous glands along the body, and the glandular structure, have not received attention. In addition, fossoriality, a fundamental trait of caecilian biology has not been considered when analyzing glandular heterogeneity and distribution. Amphibian granular glands in general produce a serous secretion that varies substantially in composition among species, though virtually always toxic9. Poison glands are present in all amphibian orders and have nor- mally been associated with defense against predators1,24. In some anurans and salamanders such glands enlarge and accumulate in certain parts of the skin, forming macroglands, constituting prominent structures in relation to the body surface9,12,25–27. In S. annulatus, although the size of the poison glands varies throughout the body, the morphological and histochemical characteristics remain the same. Additionally, the morphology of the granular glands of S. annulatus is similar to that of salamanders and newts, showing a multicellular arrangement, diferent from that of anurans, which form syncytia. Little is also known in relation to the chemical composition and activity of caecilian cutaneous secretions. Haemolytic activity was reported for Siphonops paulensis, probably as a function of a lysine-like protein28–30. Sawaya23 reported cardiotoxic activity in the cutaneous secretion of Siphonops annulatus, also demonstrating its paralyzing and lethal potential. In addition to these observations, it is known that the secretions of S. annulatus can cause strong irritation to the eyes and nasal mucosa (personal observations). We verifed the presence of proteins in the secretion of both body ends of S. annulatus, although proteins were more abundant in the secretion extracted from the posterior portion. Secretion extracted from the head (with a prevalence of mucous glands) and that from the posterior region (with prevalence of poison glands) are quite diferent, each one presenting exclusive proteins. Diferences in composition of the two extremities were also

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Figure 4. Biochemical characterization of the secretion extracted from the head and posterior region of Siphonops annulatus. (a) SDS-PAGE of the secretion of the head (H) and of the posterior region (P) (See Fig. 4S for the original image). Te numbers on the lef refer to the molecular mass markers (kDa) shown in the lef column. Main diferences between the two types of secretion are indicated by arrows. (b) C18-RP-HPLC profles of secretions extracted from the head (red) and from the posterior region (black). Te insert represents a high magnifcation of the image.

evidenced by chromatography. Te secretion of the posterior region is much more diverse and abundant when compared to the secretion extracted from the head. Tese data corroborate the observations of Sawaya23 who found greater lethality in the “milky” secretion from the skin of the tail. Siphonops annulatus have the same diameter along the body (except for the smaller head), perfectly ftting the diameter of their tunnels, which allows their blunt posteriors to block the tunnel. Phragmosis, a defensive method consisting of blocking the entrance of a hole using part of the body31 has been reported for insects31, anurans (Corythomantis greeningi32, and even mammals such as the pink armadillo Chlamydophorus truncatus33. In the case of the tree frog C. greeningi, there is a strong relationship between the head, which is used in phragmosis, and the presence of poison gland accumulations32,34. Similar to the head of this tree frog, the posterior portion of S. annulatus has internalized glandular accumulations that are arranged in honeycomb architecture, resembling the typical parotoids of toads15,27,35. However, diferent from toads, protuberant glandular accumulations would not be evolutionarily favored in animals moving within tunnels such as caecilians. Te study of the structure and function of caecilian cutaneous glands has evolved since the works of A. Sawaya22 and P. Sawaya23 studying Siphonops annulatus. Sawaya22 showed that the posterior granular glands referred to as “giant glands” by Sarasin and Sarasin20) are found in the dorsal region of the last rings, close to the cloaca, decreasing in number towards the head. However, Sawaya22 did not suggest any possible reason for such glandular distribution. Considering specifcally the mucous glands, we found in S. annulatus two basic glandular varieties (Type I and Type II) which difer in size and composition of their secretions. Tis fnding amplifes previous reports22 that observed only one type of mucous gland in this species. Here we demonstrate that Type I mucous glands produce lipid secretion in much greater amounts than Type II mucous glands. Type I glands are larger than Type II and accumulate in the head region, diminishing in size and frequency along the body. Mucous secretions, with the addition of lipid substances, may be efcient in reducing friction between the skin and the soil especially during the initial phase of burrowing, but also as the animal moves through the tunnels. In fact, we have observed in the feld that the tunnels built by S. annulatus are always lined with a shiny and slippery secretion. Gabe36 studying Ichthyophis glutinosus skin morphology, suggested that the cutaneous secretion in general could be used in fosso- rial locomotion without describing any particular glandular distribution along the body. In addition to the abundance of mucous glands, the head skin of Siphonops annulatus shows the presence of anchor spots of the dermis entering little pits in the skull bones. Tese anchor spots would maintain cohesion between the skin and the bones of the head during the constant friction between the head and the substrate when the animals move within the tunnels or when beginning excavation. Trough the images showed by Wilkinson et al.37 it can be observed that such anchor spots are quite common in the skull of several other caecilians. Te morphological and biochemical aspects of the skin and cutaneous secretion of Siphonops annulatus pre- sented in this work can be clearly related to the fossorial environment in which these animals live. While the funnel-form head mechanically opens the way through the soil, the lubricant mucous/lipid secretion produced by the mucous glands is spread over the body promoting efcient “diving” underground. In the posterior region, the granular glands form an internalized, non-protuberant macrogland, providing a defensive chemical mechanism against predators. At the same time, the poisonous body terminus can be used as a mechanical barrier, blocking the tunnel and preventing invasion by co-specifcs or potential predators.

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Material and Methods Animals and extraction of cutaneous secretion. Eight adult Siphonops annulatus (375–452 mm, mean 410 ± SD 25 mm) were collected in Ilhéus (BA) and maintained in the animal house of the Laboratory of Cell Biology of Instituto Butantan. Crude skin secretions from all specimens were collected from both head and pos- terior regions of the body (secretions from each body region were pooled for all specimens). Each extremity was separately submerged in ultrapure water poured in a Petri dish, and the skin was stimulated to release secretion by gently brushing with a sof toothbrush. Te resulting secretions were lyophilized and kept at −20 °C. One month afer secretion collection, the animals were sacrifced using lethal doses of thiopental (30 mg/Kg) and preserved in 4% Bouin fxative or 10% bufered paraformaldeyhyde (pH 7.2) for 24 h. All procedures were approved by the Ethics Committee for the Use of Animals of Instituto Butantan (CEUAIB, 174/2004 e 444/2008) and all methods were performed in accordance with the relevant guidelines and regulations.

Anatomical study. Following sacrifce, the head and the posterior portion of two individuals were sepa- rated from the body. In each extremity, tangential sections to the skin were made in order to expose deeper skin layers where the glands are present. Subsequently, the exposed area was cleaned with a toothbrush, removing the glandular content. In order to examine the skull, the head was cleaned of tissue by successive washings in sodium hypochlorite. Samples were analyzed and photographed with a Leica M205-A stereomicroscope using the sof- ware LAS (Leica ). ® Te skin of the® head and the posterior portion of two other preserved specimens was tangentially sectioned as described above. Heads and posterior regions were then dehydrated in a critical point dryer, sputter coated with gold, and examined under a scanning electron microscope FEI Quanta 250, operating at 10 kV.

Histological study. Afer fxation, four samples of dorsal skin were removed along the body of four individ- uals. Head and posterior portions were removed and decalcifed in 4% EDTA solution, pH 7.2, for two months. All samples (dorsal skin fragments and decalcifed heads and posterior portions) were embedded in parafn or historesin Leica . Sections 2−5 μm thick were stained with haematoxylin-eosin and toluidine blue-fuchsin. Histological® sections were stained as follows: bromophenol blue for identification of proteins, periodic acid-Schif (PAS) for identifcation of carbohydrates in general, alcian blue pH 2.5 for identifcation of acidic carbohydrates, and Sudan black for identifcation of lipids38. Slides were photographed with an Olympus BX51 microscope using Image Pro Express sofware.

Electrophoresis (SDS-PAGE) and chromatography (RP-HPLC). Lyophilized aliquots of secretion from the head and posterior region were dissolved in phosphate bufered saline and quantifed by spectro- photometry using NanoVue Plus. Protein composition of skin secretions from the two areas was evaluated by electrophoresis in 12% polyacrylamide gel (PAGE) containing sodium dodecyl sulfate (SDS) under reducing conditions39. Te gel was then stained with silver. Crude secretions from head and the posterior regions were analyzed by reverse-phase liquid chromatography (RP-HPLC) using a binary HPLC system (20 A Prominence, Shimadzu Co., Japan). Te sample was loaded on a C18 column (Phenomenex C18, 5 μm, 100 Å, 250 mm × 1 mm) and the content was eluted by a two-solvent sys- tem: (A) acetic acid (AA)/ H2O (1:999) and (B) AA/CAN/H2O (1:900:99) in a 0–90% gradient of solvent B over 20 min, afer 5 min isocratic elution with 0% B. Te fow rate was constant, set at 0.2 mL/min−1 at an oven temper- ature of 30 °C. Te elutes were monitered by a Shimadzu SPD-M20A PDA detector scanning from 200 to 500 nm.

Data Availability. Te datasets generated during the current study are available from the corresponding author on reasonable request. References 1. Duellman, W. E. & Trueb, L. Biology of Amphibians. (McGraw-Hill, 1996). 2. Himstedt, W. Die Blindwuhlen. (Westarp Wissenschafen, 1996). 3. Pough, F. H. et al. Herpetology. 4th ed. (Sinauer Associates, 2016). 4. Nussbaum, R. A. Caecilians in Encyclopedia of Reptiles and Amphibians (eds Cogger, H. C. & Zweifel, R. G.) 52–59 (Academic Press, 1998). 5. Jared, C., Navas, C. A. & Toledo, R. C. An appreciation of the physiology and morphology of the caecilians (Amphibia: Gymnophiona). Comp. Biochem. Physiol. 123, 313–328 (1999). 6. Gomes, A. D., Antoniazzi, M. M., Navas, C. A., Moreira, R. G. & Jared, C. Review of the reproductive biology of caecilians. S. Am. J. Herpetol. 7, 191–202 (2012). 7. Frost, D. R. Amphibian species of the world: an online reference. Version 6.0 (10/17/2017). http://research.amnh.org/herpetology/ amphibia/index.html (2017). 8. Fox, H. Te skin of Ichthyophis (Amphibia:Caecilia): an ultrastructural study. J. Zool. Lond. 199, 223–248 (1983). 9. Toledo, R. C. & Jared, C. Cutaneous granular glands and amphibian venoms. Comp. Biochem. Physiol. 111, 1–29 (1995). 10. Taylor, E. H. Te Caecilians of the World. (University of Kansas Press, 1968). 11. Delfno, G., Nosi, D. & Giachi, F. Secretory granule-cytoplasm relationships in serous glands of anurans: ultrastructural evidence and possible functional role. Toxicon 39, 1161–1171 (2001). 12. Mailho-Fontana, P. L. et al. Parotoid, radial, and tibial macroglands of the frog Odontophrynus cultripes: diferences and similarities with toads. Toxicon 129, 123–133 (2017). 13. Brodie, E. D. Jr. & Gibson, L. S. Defensive behavior and skin glands of the northwestern salamander. Ambystoma gracile. Herpetologica 25, 187–194 (1969). 14. Brodie, E. D. Jr. & Smatresk, N. L. Te antipredator arsenal of fre salamanders: spraying of secretions from highly pressurized dorsal skin glands. Herpetologica 46, 1–7 (1990). 15. Mailho-Fontana, P. L. et al. Passive and active defence in toads: the parotoid macroglands in Rhinella marina and Rhaebo guttatus. J. Exp. Zool. 321, 65–77 (2014). 16. Regis-Alves, E. et al. Structural cutaneous adaptations for defense in toad (Rhinella icterica) parotoid macroglands. Toxicon 137, 128–134 (2017). 17. Leydig, F. Lehrbuch der Histologie. (Verlag von Meidinger Sohn, 1857).

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